Biology subjects

Takahashi, M. K.

Publications and source records attributed to Takahashi, M. K..

3 recordsLinked to original sources

bioRxiv2026

Disruption of sRNA Function Using Synthetic Arginine Rich Motif Peptides

Small RNAs (sRNAs) regulate the expression of many genes including those involved in antibiotic resistance and bacterial virulence, making them potential therapeutic targets. A molecule that binds an sRNA could interfere with its ability to bind its target mRNA and disrupt the regulation mechanism. Randomization and screening of natural arginine rich motif (ARM) peptides led to peptides capable of interfering with the sRNA MicF's ability to regulate ompF in Escherichia coli. Molecular dynamics simulations suggested that this effect was not a result of a direct disruption of the MicF-ompF interaction. Instead, the peptides interfere with binding of the chaperone Hfq, which is required for MicF-mediated regulation. Subsequent testing demonstrated peptide specificity for MicF over two other Hfq scaffolds and the ability to disrupt regulation of two additional MicF targets. Together, these findings support the use of synthetic ARMs as a potential tool for modulating sRNA function in bacteria.

synthetic biology↗

bioRxiv2024

Beyond membrane permeability: A role for the small RNA MicF in regulation of chromosome replication and partitioning

Small regulatory RNAs (sRNA) have been shown to play a large role in the management of stress responses in Escherichia coli and other bacteria. sRNAs act post-transcriptionally on target mRNA through an imperfect base pairing mechanism to regulate downstream protein expression. The imperfect base pairing allows a single sRNA to bind and regulate a variety mRNA targets which can form intricate regulatory networks that connect different physiological processes for the cells response. Upon exposure to antimicrobials and superoxide generating agents, the MicF sRNA in E. coli has been shown to regulate a small set of genes involved in the management of membrane permeability. Currently, it is unknown whether MicF acts on other processes to mediate the response to these agents. Using an sRNA interaction prediction tool, we identified genes in E. coli that are potentially regulated by MicF. Through subsequent analysis using a sfGFP-based reporter-gene fusion, we have validated two novel targets of MicF regulation: SeqA, a negative modulator of DNA replication, and ObgE, a GTPase crucial for chromosome partitioning. Importantly, the interaction between MicF and these target mRNAs is contingent upon the presence of the RNA chaperone protein, Hfq. Furthermore, our findings affirm the role of MicFs conserved 5 seed pairing region in initiating these regulatory interactions. Our study suggests that, beyond its established role in membrane permeability management, MicF exerts control over chromosome dynamics in response to distinct environmental cues, implicating a more multifaceted regulatory function in bacterial stress adaptation.

microbiology↗

bioRxiv2023

Identifying antisense oligonucleotides to disrupt small RNA regulated antibiotic resistance via a cell-free transcription-translation platform

Bacterial small RNAs (sRNAs) regulate many important physiological processes in cells including antibiotic resistance and virulence genes through base pairing interactions with mRNAs. Antisense oligonucleotides (ASOs) have great potential as therapeutics against bacterial pathogens by targeting sRNAs such as MicF, which regulates outer membrane protein OmpF expression and limits permeability of antibiotics. Here, we devise a cell-free transcription-translation (TX-TL) assay to identify ASO designs that sufficiently sequester MicF. ASOs were then ordered as peptide nucleic acids conjugated to cell-penetrating peptides (CPP-PNA) to allow for effective delivery into bacteria. Subsequent minimum inhibitory concentration (MIC) assays demonstrated that simultaneously targeting the regions of MicF responsible for sequestering the start codon and the Shine-Dalgarno sequence of ompF with two different CPP-PNAs synergistically reduced the MIC for a set of antibiotics. This investigation offers a TX-TL based approach to identify novel therapeutic candidates to combat intrinsic sRNA-mediated antibiotic resistance mechanisms.

synthetic biology↗